For high-level observations of gene expression within tissue, intracellular localization, and cell morphology, a pretreatment (clearing treatment) for increasing the light-transmitting property of tissue has been performed with use of a clearing reagent in recent years. Various clearing reagents and pretreatment methods have thus been developed.
Non Patent Literatures 1 and 2 each disclose a clearing treatment technique involving use of benzylbenzoate/benzylalcohol (BABB method). The BABB method is a classic tissue clearing method involving use of an organic solvent, and has been applied as a method for preparing a cleared sample to be observed through a light-sheet macro-zoom microscope (see, for example, Non Patent Literature 3).
Non Patent Literature 4 discloses a technique (SeeDB method) of adjusting the refractive index with use of fructose. The SeeDB method is primarily intended to minimize a change caused to biological tissue during the treatment, and thus the biological tissue substantially completely maintains the molecular-level structure, membrane structure, axon shape, and the like. The SeeDB method, in a case where it uses a two-photon microscope and an optimized lens, allows observation at a level of the whole brain.
Non Patent Literature 5 discloses a clearing technique (CLARITY method) based on a physicochemical method involving use of an electrophoresis apparatus. The CLARITY method uses an electrophoresis apparatus to physicochemically treat brain tissue packed with use of an acrylamide polymer for removal of lipid components to make the brain tissue transparent. The CLARITY method allows high-level clearing in approximately two weeks in a state where the molecular-level structure of tissue is maintained. The CLARITY method is applicable to both (i) fluorescent protein labelling and (ii) immunostaining.
Non Patent Literature 6 discloses a technique (Scale method) that uses a water-soluble reagent to successfully minimize quenching of fluorescence signals.